The eukaryotic genome is organized into domains. The higher order chromatin structure of each domain as well as the replication timing of each domain is independent of that of the adjacent domains. Such compartmentalization of the genome implies the existence of boundaries that insulate one domain from another. We have analyzed the 5' boundary region of the chicken beta-globin domain in an attempt to locate the insulator activity. We have identified a 1.2kb fragment in the 5' boundary region that is capable of insulating a reporter gene from the activating effects of a nearby mouse beta-globin Locus Control Region (LCR) in human erythroleukemic cell line K562. From deletion analysis, we find that most of the insulating activity is concentrated in a hypersensitive site (5' HS4) within the 1.2kb fragment. This DNase 1 hypersensitive site is GC rich and contains a number of binding sites for several unknown factors and one known factor Sp1. Interestingly, this 5' most hypersensitive site is also present in mouse and human beta-globin domains. In transgenic Drosophila, the chicken beta-globin insulator protects the "white" minigene from the chromatin structure and regulatory elements near the site of integration. The action of the insulator thus is not restricted to erythroid or mammalian cells, suggesting that such elements may serve an important and widely distributed function in the organization of chromatin structure.